CTG18.1-mediated Fuchs Endothelial Corneal Dystrophy: defining unique signatures of transcriptomic dysregulation

Abstract

Purpose : Fuchs endothelial corneal dystrophy (FECD) is a common degenerative disease predominantly caused by an expanded (≥ 50 copies) CTG repeat (termed CTG18.1) in an intron of the transcription factor encoding gene TCF4. CTG18.1-mediated FECD is unique among repeat expansion diseases in its high frequency among the general population and a pathophysiology limited to corneal endothelial cells (CECs). Here we generated transcriptome data from primary CEC cultures to identify the differentially expressed transcripts that may represent key biomarkers to enhance understanding of the pathophysiology of FECD.

Methods : We extracted total RNA from primary CEC cultures from 10 unrelated individuals including: 4 unaffected controls, 3 CTG18.1-expansion negative FECD (NE-FECD) and 3 CTG18.1 expansion-positive (E-FECD) patients. RNA-seq libraries were prepared using oligo(dT) beads to enrich for poly-A mRNA and sequenced in accordance with standard Illumina paired-end protocols. Differential gene expression was assessed via DeSeq2 and adjusted with independent hypothesis weighting, while differences in pre-mRNA splicing were analyzed via IsoformSwitchAnalyzeR to identify canonical isoform variants.

Results : Of the 4,497 genes significantly (adj. p-value ≤ 0.05) differentially expressed in E-FECD, 1,248 were unique to E-FECD when compared with NE-FECD with 486 having a log2-fold difference of at least 1. Among these 486 genes, ontology analysis revealed enrichment of genes involved in signaling pathways, cell structure, extracellular matrices, and protein synthesis. Isoform analysis revealed 303 genes with significant differences in isoform expression between E-FECD and controls, 205 of which are predicted to produce a functional difference due to the inclusion or exclusion of a functional exon. Included within this dataset are genes with known MBNL1 splicing regulation, suggesting that splicing dysregulation could be due to sequestration of splicing factors by the CTG18.1-expanded transcript.

Conclusions : A comparison of transcriptomic profiles of control, E-FECD and NE-FECD provides a dataset of differentially expressed transcripts and aberrantly regulated pre-mRNA splicing events that uniquely occur in E-FECD. These data provide insight into disease mechanisms and highlight targets for CTG18.1 expansion-specific translational interventions.

This is a 2021 ARVO Annual Meeting abstract.